Microchannel devices are finding increased use in the identification and synthesis of chemical and biological species. Employing transverse channel dimensions from a few microns to about one millimeter, such systems may permit the miniaturization and large-scale integration of many chemical processes in a manner analogous to that already achieved in microelectronics. Applications for microchannel devices now under development include such diverse processes as DNA sequencing, immunoassay, the identification of explosives, identification of chemical and biological warfare agents, and the synthesis of chemicals and drugs.
Most microchannel systems for chemical and biological analysis employ some variant of electrochromatographic or electrophoretic separation. In chromatographic processes, bulk electroosmotic motion of a fluid is induced by applying an electric field along the length of the separation column. Individual species move through the column at various speeds due to preferential adsorption on stationary surfaces such as the channel walls or an internal porous packing. In contrast, electrophoretic processes involve little or no bulk fluid motion. Here the applied electric field instead produces motion of ionic species through a stationary or nearly stationary transport medium that may be either a fluid or a gel. Species separation in electrophoretic processes occurs as a result of differing ratios of the ion charge to the ion mobility and consequent differing ion speeds.
Electroosmotic flows offer two important benefits over pressure-driven flows for transport processes in microchannel devices. First, transport speeds in electroosmotic flow are independent of the width and depth of a channel cross-section over a wide range of conditions, making this technique for driving fluid motion extensible to extremely small physical scales. In contrast, pressure-driven flows require a pressure gradient that increases inversely with the square of the minimum transverse dimension to maintain a given fluid speed. Second, the profile of the fluid velocity across the cross-section of a long straight channel is essentially flat in electroosmotic flows, again over a wide range of conditions. All transverse variation in the axial speed is confined to a small region adjacent to the channel walls and comparable in thickness to the electric Debye layer. The benefit of this flat velocity profile is that samples may be transported over long ranges with very little hydrodynamic dispersion due to nonuniform fluid speeds.
Electrophoretic processes offer somewhat different benefits in microchannel devices. Because the overall dimensions of microchannel devices tend to be only a few centimeters, very large electric fields may be produced by relatively small electric potentials. This permits larger ion speeds and reduces the overall time for separation processes. Electrophoretic motion is also easy to produce in microchannel systems since, like electroosmotic flow, only electrodes and a power supply are required to produce the phenomenon. Mechanical pumps are unnecessary. Also like electroosmotic flows, this method of producing species motion introduces little extraneous spreading of a sample band as it moves along the separation column since the electric field in a long straight channel is spatially uniform.
Despite these benefits in long-range transport, electrokinetic mechanisms are not particularly well suited to producing a thin sample band for subsequent processing in a separation or other process channel. The reason for this is that species motion in both electroosmotic and electrophoretic transport processes is governed by the highly-diffusive Laplace equation, and the region over which motion is induced usually occupies at least one channel width. As a result, the thickness of a sample band produced by such motion will usually exceed the channel width; this is not acceptable for many processes of practical interest.
For electrochromatographic and electrophoretic separation processes, the thickness of the initial sample band measured in the direction of the sample motion may need to be small compared to the channel width. Separation columns in microchannel systems are typically not very long, so final spacings between constituent bands may span only a channel width or so. To resolve these bands requires that the thickness of the sample band initially injected into the column is much smaller than the band spacing or, equivalently, much smaller than the channel width. In addition to separation processes, small sample sizes and sharp definition of species interfaces are also desirable during routine sample transport. These desired characteristics allow more precise control over processes such as mixing, dilution and synthesis.
The invention described here provides a method and apparatus for producing a sample band for subsequent processing wherein the thickness of the band in the direction of sample motion is small compared to the channel width. Further, the thickness of the band may be controlled to provide a desired sample volume or size. This method can even produce sample bands having an overall thickness an order of magnitude or so smaller than the channel width. The method can be implemented using conventional channel geometries and conventional electrical hardware. Additional benefits can be obtained by using these new methods in conjunction with improved channel geometries. Such improved geometries are also disclosed here.
FIG. 1 schematically illustrates a very simple microchannel system 100 for chemical analysis. Here, the channels are fabricated on a planar substrate 101. Reservoirs 102-105 have access ports (not shown) that permit introducing and extracting fluid through the top or bottom faces of the substrate. Channels 112-115 are filled with a fluid or gel-material hereinafter referred to as a xe2x80x9ctransport medium,xe2x80x9d which supports migration of ions or charged particles of a sample material either through or with the transport medium under the influence of an applied electrical field. The process channels 112-115 may also contain a separation matrix comprising a porous or granular material, a microfabricated pattern of obstacles, or a plurality of protrusions that promote species separation. The reservoir access ports (not shown) may also be used to control the hydrostatic pressures in the reservoirs or they may be left open to maintain reservoir pressures equal to the atmospheric pressure. Similar access holes (not shown) are used to insert electrodes 106-109 that are connected to power supply 110 through leads 106xe2x80x2-109xe2x80x2 respectively.
Power supply 110 is used to apply electric fields along one or more of channels 112-115 emanating from junction 111. The electric field is the negative of the gradient of the electric potential. The overall electric field is applied by controlling the differences in electric potential between the reservoir electrodes or, equivalently, by controlling the electric current flow to the individual electrodes. Although the electric field within the junction region is generally multidimensional, the electric field and induced sample transport is nearly one dimensional and uniform along straight channel segments, provided that the Debye layer thickness is small compared to the transverse channel dimensions. Under this restriction, easily met by most practical systems, the electrokinetic transport speed is simply proportional to the electric field at any point within the channels or junction. Thus, control of the electric field is equivalent to controlling the sample transport speed.
In a separation process, different species within a sample band (not shown) move along process channel 112 (or separation column) at different speeds due to differences in surface adsorption or differences in ion charge and mobility. As a result, the sample separates into a series of constituent bands that are detected as they pass through a detection device 120, located toward the end of process channel 112. The contents of the sample are inferred from the observed arrival times of the separated bands. Since the measurement resolution depends on physical separation between the bands, it is desirable that the bands be as distinct and sharply defined as possible.
The spacing between constituent bands is generally proportional to the length of the separation column. Because microchannel devices are intended to be small, separation columns in these devices tend to be short by conventional standards and rarely exceed several centimeters. As a result, the spacing between constituent bands at the detector location may be only a fraction of the channel width. Because band spacings less than the initial thickness of the sample cannot be resolved as distinct entities, the initial thickness of the band injected into the process channel must also be small compared to the channel width. More generally, the thickness of the injected band must be as small as possible to provide maximum resolution for all band spacings, while at the same time providing just sufficient sample volume for accurate down-stream detection.
To produce a sample band and inject it into process channel 112 of system 100, the sample material is first introduced into the lower supply channel reservoir 103 and then transported through supply channel 113 by applying an electric field along supply channel 113 and waste channel 114. This field is applied by using the power supply to impose a potential difference between the electrode 107 in the supply reservoir 103 and the electrode 106 in the waste reservoir 102 terminating waste channel 114. During this step of the process, the power supply prevents any current flow to electrodes 109 and 108 located in the buffer and process reservoirs 104 and 105, respectively, so as to prevent any significant transport along the buffer and process channels, 115 and 112. After completing this step, the junction 111 is filled with sample material (not shown). This volume of sample material contained within the junction is then transported into the process channel 112 to form the sample band. The sample transport during this step is induced by applying an electric field along buffer and process channels 115 and process channel 112 using the buffer and process channel electrodes 109 and 108 respectively. During this step the power supply prevents current flow along the supply and waste channels. However, because the electric field lines tend to bulge from the primary channel into the ends of the supply and waste channels, sample material is removed from the supply and waste channels, forming a tail on the sample band as it is transported along the process channel. As a result, a sample band produced in this manner generally has a thickness that exceeds the channel width.
In order to illustrate the process and to promote an understanding of the invention a series of illustrations follows. The schematic diagrams of sample motion, shown in FIGS. 2-9, are based on a series of detailed computational simulation using a variety of conditions and parameters beginning with the prior art configuration. The computational model used to perform the numerical simulations is described below in the section entitled xe2x80x9cDetailed Description of the Invention.xe2x80x9d The actual graphical output of each of the computer simulations represented by FIGS. 2-9, is shown in the corresponding FIGS. 12-19. As will be explained below, the model and the computed results are equally applicable to sample transport by electroosmotic or electrophoretic means or to any combination of these two transport mechanisms.
FIG. 2 schematically illustrates the sample transport for the conventional two-step process (described above) used to produce a sample band 200 and inject it into a process channel 202. The first step of this process is shown in the upper sequence of frames comprising FIGS. 2A-2D. These frames illustrate the behavior of flow through channels 201-204 at four succeeding instants in time. Time increases from left to right, with each frame later than the preceding frame by a discrete increment of time, ti The lower sequence of four frames (FIGS. 2E-2H) illustrates the second step of the process, again read from left to right. The first frame, FIG. 2E, of the second step (second row) is identical to the last frame, FIG. 2D, of the first step (first row). For the sake of clarity and to promote a better understanding of the invention, only the channel segments near the junction 205 are shown. Furthermore, those regions within the channel which are occupied by sample material are illustrated as comprising a plurality of black dots, analogous to tracer particles that are carried along with the sample, again for clarity.
In the first step, (FIGS. 2A-2D) sample material 206, represented by black tracer particles, is transported from supply channel 204 through junction 206 and along the waste channel 201 by applying an electric potential between the supply and waste reservoirs (not shown). These potentials establish an electric field that drives sample transport along the supply channel, through the junction, and into and waste channel. The direction of fluid motion, or species migration, is indicated by arrows 208. Unintended lobes 207 of sample material 206 also penetrate about one channel width into process channel 202 and buffer channel 203. This penetration results from bulges in the electric field lines extending into the process and buffer channels, 202 and 203 respectively, not from diffusion. Once the junction 205 is completely filled, as in FIGS. 2C and 2D, the first step of this conventional process is terminated by removing the applied electric field.
As noted above, the second step in the conventional two-step process is illustrated in the sequence of frames FIGS. 2E through 2H. Here, an electric potential is applied between the reservoir electrodes (not shown) terminating buffer channel 203 and process channel 202 to produce sample motion to the right, indicated by arrow 208 in FIG. 2F, injecting a sample band into the head of process channel 202. However, as seen in the last two frames (FIGS. 2G and 2H), the thickness, d, of sample band 200, is more than twice the channel width. In addition sample band 200 is even further elongated by long trailing tails 200xe2x80x2. These tails result from bulges in the electric field lines now extending into the waste and supply channels, 201 and 204. Since the electric field is relatively weak in side lobes 207, sample material 206 is released very slowly from the vertical supply and waste channels into the process channel 202. The end result is an injected sample band 200 having an effective thickness of three to four channel widths.
To reduce the thickness of injected samples and to help eliminate sample tails, improved methods of sample manipulation have been explored in the prior art. FIGS. 3A-3H illustrate an improved prior-art two-step method used in the prior art to produce and inject a sample band. This improved method reduces the thickness, d, of the sample band 200 and largely eliminates the long trailing tails 200xe2x80x2 shown earlier in FIGS. 2A-2H. The first step in this method is the same as shown previously; the top set of frames, FIGS. 3A-3D, are identical to those in FIGS. 2A-2D. The sample material 206 is first transported along the supply channel 204, through the junction 205 and then toward the waste channel reservoir (not shown). The lower frames, FIGS. 3E-3H, show subsequent transport of the sample band 200 into the process channel 202. In contrast to FIGS. 2A-2H, the electric potentials of FIGS. 3A-3H are applied to the four reservoir electrodes (not shown) in a manner that causes transport (shown as arrows 208 in FIG. 3F) from the buffer channel 203 into the supply 204, waste 201, and process 202 channels, injecting sample band 2003 into the process channel 202. In the last two frames, FIGS. 3G-3H, the sample band is moving from left to right along the process channel toward a detector (not shown), while other portions of sample material are moving from the junction 205 toward both the supply and waste reservoirs (not shown).
In the second step of the process, FIGS. 3E-3H, the field is applied by setting the electrode potential of the buffer reservoir above the potential of each of the other three reservoirs. In general_however, the required polarity of the applied field depends upon the sign of the Zeta potential in electroosmotic flows while in electrophoresis the required polarity depends on the sign of the charge on the ion species comprising the sample. In the particular example shown in FIGS. 3E-3H, the magnitude of the applied electric field in the waste, process and supply channels, 201, 202, 204 respectively, is the same. However, this need not be the case, in general, provided that the mean transport speeds in supply and waste channels 204 and 201 are a significant fraction of the mean transport speed in process channel 202.
The improved method shown in FIGS. 3A-3H is capable of reducing the thickness, d, of the resulting sample band 2003 by about a factor of two. This limitation is based on the simple observation that about half of the sample volume in junction 205 at the beginning of the second step (FIG. 3E) is ahead of the center of the junction. Thus, even if transport speeds in the supply and waste channels 204 and 201 are increased dramatically during this step, it is not possible to reduce the effective sample thickness, d, to less than about one channel width. However, this approach does effectively eliminated the long tails 200xe2x80x2 that would otherwise follow sample band 2003 into process channel 202.
Recognizing the need for further reduction in the thickness of sample bands, Jacobson and Ramsey (Anal. Chem. 1997, 69, 3212-3217) proposed a sample focusing technique which they have demonstrated in computational and experimental studies. Their basic concept, as it relates to preparation of sample bands is illustrated in FIGS. 4A-4H. For convenience, the discussion immediately below uses language appropriate to electroosmotic flow, though the same procedure is also applicable to sample transport by electrophoresis.
The first step of the process, shown in FIGS. 4A-4D, illustrates the geometry of the focusing apparatus and the first step in this method. The apparatus consists of two connected junctions 205 and 209; the upper junction 205 plays the same role as those in previous figures while the lower junction 209 is for focusing the sample material 206.
During the first step, shown in FIGS. 4B and 4C, sample material 206 enters the lower focusing junction 209 from below, through the supply channel 204, while a buffer fluid enters junction 205 from both the left and the right in opposite (countervailing) directions, through focusing channels 210 and 211. Flow directions in the vicinity of junction 209 are shown as arrows 208. The incoming buffer streams from the focusing channels 210 and 211 enter focusing junction 209 to restrict and confine the width of the sample stream 206 as it moves toward the upper junction 205 thereby forming focused sample stream 206xe2x80x2 so that it occupies only a fraction of the width of supply channel 204, in this case about one-third of that width. As before, focused sample stream 206xe2x80x2 has bulged slightly toward both process and buffer channels 202 and 203, producing reduced lobes 207xe2x80x2. Here, the extent of bulging is greatly reduced because the focused sample stream 206xe2x80x2 crosses the upper junction 205 very near to the centerline of the supply and waste channels 204 and 201. The width and the position of a focused sample stream 206xe2x80x2 are generally controlled by the relative fluid speeds in left, right and lower channel legs 210 and 211 of the focusing junction 209. Focusing thus offers a means of controlling the thickness, d, of a sample band 2004 without altering the channel geometry.
FIGS. 4E-4H illustrate step 2 of this improved method, whereby a portion of the focused sample stream 206xe2x80x2 is injected into process channel 202 to produce the final sample band 2004. Note, that the lower focusing junction 209 is omitted from these lower frames. At the beginning of this step, the focused sample 206xe2x80x2 has already been transported through junction 205. The sample band is formed by raising the electric potential in the buffer channel reservoir (not shown) relative to that of reservoirs terminating the supply, waste and process channels (not shown). This step is therefore the same as the second step of the method shown earlier in FIGS. 3E-3H, with the exception that here the initial thickness of the sample stream 206 has been reduced to produce focused sample stream 206xe2x80x2 using a focusing junction 209 as described above. We see in FIGS. 4F and 4G that the thickness, d, of the resulting sample band 2004 is not substantially altered during the final step. We also see that the thickness, d, of the resulting sample band is greatly reduced relative to that produced by the methods previously illustrated in FIGS. 2 and 3.
Although the sample focusing method described above and shown in FIGS. 4A-4H can produce a thin sample band 2004, and provides a means for controlling the sample band thickness, d, it does require the use of auxiliary channels 210 and 211, junction 209, additional buffer reservoirs (not shown), and added electrodes, fill ports, and electrical connections (not shown), in order to perform the focusing process.
In summary, the simplest prior-art process produces a sample band thickness of at least two to three channel widths (FIGS. 2A-2H). This can be reduced to about one channel width by inducing transport away from the junction into the supply and waste channels as the sample band is transported into the process channel (FIGS. 3A-3H). Another procedure of prior art, known as sample focusing, is effective at producing a sample band having a thickness that is small compared to the channel width (FIGS. 4A-4H). However, this approach requires an additional focusing junction and additional channels, reservoirs, electrical connections and controller channels.
To overcome the shortcomings of the prior art, the present invention provides a new method for producing sample bands having a controlled thickness that may be far less than the widths of the channels. The new approach comprises two primary steps: (1) inserting an initial sample band into one leg of a junction and (2) thinning of the initial sample band by transporting it across the junction one or more times using a diverging flow field within the junction to stretch and thin the sample. The first step may be performed by one of the prior art methods already shown in FIGS. 2A-2D or 3A-3D or, alternatively, by a new method, unique to the present invention, that substantially reduces the thickness of the inserted sample band. The second step, that of thinning the sample band, is also unique to the present invention. This thinning step may comprise a number of steps or repetitions in which the sample is transported back and forth across the junction. At the end of the final thinning step, the sample band is injected into the head of the process channel, ready for subsequent processing.
An important advantage of all embodiments of the present invention is that they rely only on altering the sequence of applied electric fields to control the volume and thickness of the sample band. The required microchannel hardware comprises simply a junction formed at intersecting channels and a power supply to control the electric fields in each of the channels. The geometries of the junction and channel network are not critical and need not be altered in any way from existing or conventional designs. In particular, there is no need for channels of varying or differing depth. Of course, alterations to the channels and junction can be made to optimize these methods, and the channels abutting the junction may have the same or differing widths. However, with or without these geometric alterations, the present methods can be easily implemented using existing technology and existing hardware.
The present methods can be used to reduce the injected sample thickness by any desired degree, provided that the transport speeds used during sample manipulation are sufficiently high. The use of these high speeds reduces the extent of molecular diffusion of the sample band during manipulation. Finally, these new methods may be employed using multiple junctions distributed along a single buffer/process channel to inject multiple sample bands into one process stream. They may also be employed using multiple buffer/process channels to simultaneously inject one or more samples into each.
The present invention, therefore, provides a simple means for reducing and controlling the thickness of injected sample bands and does not require additional focusing channels, reservoirs or electrical hardware to accomplish this result.
The new method of producing a thin sample band in a microchannel system comprises a series of steps in which the sample is manipulated by controlled transport through a junction. The junction is formed at the intersection of two primary channels, the buffer and process channels, running in one direction (the primary axis) and a pair of side channels, supply and waste channels, usually running in a nominally perpendicular direction (secondary axis). The first step of the new method is insertion of a sample band into one of the primary channels. The insertion step can be performed in either of the conventional fashions shown earlier in FIGS. 2 and 3, or by using a new insertion process described just below.
The first step of the new insertion procedure is to transport sample material from a supply channel through the junction and out the waste channel. In the second step of insertion, unique to the present invention, the sample volume within the junction is reduced and positioned to produce an initial sample; band. During this second step of insertion, sample material enters the junction from either the buffer or the process channel and leaves the junction along the supply and waste channels. The transport during this step is induced by applying electric fields along three of the four channels, while preventing current flow along the remaining channel containing the initial sample band at the end of the step.
After this insertion step, the thickness of the initial sample band may be reduced by one or more thinning steps, also unique to the present invention. During these thinning steps, the applied electric field induces sample transport from one of the primary channels (buffer or process) toward the other three channels. As the sample band moves across the junction, it is stretched and thinned by the diverging transport field within the junction and part of the sample band is transported into the side channels (supply and waste). In multiple thinning steps, the sample band moves back and forth across the junction, each time stopping and reversing direction before or slightly after it enters the buffer or process channels. After thinning to a desired thickness, the sample band is injected into the head of the process channel.
In addition to these new methods of sample insertion and sample thinning, the present invention also describes a new procedure for performing focusing of a sample stream and injection of a sample band within a single junction. The prior art of FIGS. 4A-4H required two junctions to perform the same task. This new method employs a three-step process comprising: a first step in which a sample stream is transported into a junction using transport from intersecting channels to focus the sample stream; a second step in which the narrow focused stream is transported in the reverse direction so that the focused portion of the sample stream extends across the full width of the junction; and a third step in which a portion of the focused stream is injected into a process channel.
In addition to these new processes, the new apparatus of the present invention provides improved junction and channel geometries that increase the thinning that occurs each time the sample band traverses the junction and reduce the bow of the final sample band. Increased thinning results from enlarging the junction beyond the bounds of the channel intersection. Bow is reduced by cusp-like extensions to the process channel walls, protruding into the junction. These extensions increase the axial sample speed along the channel walls near the channel inlet, allowing the band to enter the process channel with little bowing in the direction of motion. Similar extensions to the supply, waste and buffer channels are also beneficial for some more sophisticated methods of producing and injecting a sample band. This new apparatus may be used either with previously developed methods of sample manipulation or with the improved methods of the present invention.
The present invention, therefore, provides a method and apparatus for producing a sample band and injecting it into a process channel such that the thickness of the sample band is small in the direction of its motion but which occupies the full width and height of the channel.
Furthermore, the present invention provides a means for varying and controlling the thickness of the sample band by controlling the sequence and magnitude of electric fields applied along each channel, without modification of the channel and junction geometries.
Another object of the instant invention is to provide a new method for improving the performance of microfabricated devices used for analysis, synthesis and other processes involving chemical and biological species.
Further, by providing a means for controlling the thickness of initial sample bands, the present invention provides a means for improving the resolution of separation processes used to identify different chemical species by differences in their arrival times near the end of the process channel.
It is yet another object of this invention to provide a means for improving the resolution of separation processes or other processes in which thin, sharply defined chemical bands are desirable.
It is yet another object that this invention be applicable to both electroosmotic and electrophoretic processes driven by applied electric fields.
It is a further object of this invention that it be applied to microchannel devices comprising channels that are filled with a fluid or with a gel. In addition to these fluid and gel transport media, the transport channels may also contain a separation matrix comprising a porous or granular material or microfabricated patterns of obstacles or surface protrusions.
It is another object of this invention to provide an improved junction design comprising a cusp-like extension, or extensions, on the process channel walls, protruding into the junction, for increasing the axial transport speed of the sample band along the channel walls near the channel inlet.
FIG. 1 illustrates a typical prior art channel configuration of a microchannel system used for processing of chemical or biological samples.
FIGS. 2A-2H illustrate a conventional method for producing and injecting a sample band. The illustration is based on the simulation results shown in FIG. 12.
FIGS. 3A-3H illustrate a conventional method for producing and injecting a sample band. The illustration is based on the simulation results shown in FIG. 13.
FIGS. 4A-4H illustrate a conventional method for producing and injecting a sample band. The illustration is based on the simulation results shown in FIG. 12.
FIGS. 5A-5L illustrate a conventional method for producing and injecting a sample band. The illustration is based on the simulation results shown in FIG. 15.
FIGS. 6A-6P illustrate a conventional method for producing and injecting a sample band. The illustration is based on the simulation results shown in FIG. 16.
FIGS. 7A-7P illustrate a conventional method for producing and injecting a sample band. The illustration is based on the simulation results shown in FIG. 17.
FIGS. 8A-8L illustrate a conventional method for producing and injecting a sample band. The illustration is based on the simulation results shown in FIG. 18.
FIGS. 9A-9L illustrate a conventional method for producing and injecting a sample band. The illustration is based on the simulation results shown in FIG. 19.
FIG. 10 illustrates an improved geometry of a junction for producing a thin sample band with reduced bowing of the band.
FIG. 11A illustrates a square junction having a height and width larger than the widths of the channels.
FIG. 11B illustrates a circular junction having a diameter larger than the widths of the channels.
FIG. 12 illustrates graphical output of a computer simulation of a conventional method for producing and injecting a sample band.
FIG. 13 illustrates the graphical output of a computer simulation of an improved prior art method for producing and injecting a sample band.
FIG. 14 illustrates the graphical output of a computer simulation of an improved prior art method for producing and injecting a thin sample band by electrokinetic focusing.
FIG. 15 illustrates the graphical output of a computer simulation of the first embodiment of the present invention.
FIG. 16 illustrates the graphical output of a computer simulation of the second embodiment of the present invention.
FIG. 17 illustrates the graphical output of a computer simulation of the third embodiment of the present invention.
FIG. 18 illustrates the graphical output of a computer simulation the fourth embodiment of the present invention.
FIG. 19 illustrates the graphical output of a computer simulation of the fifth embodiment of the present invention.